Sanglifehrin A (SFA) is a recently discovered natural compound that has strong biological activity as an immunosuppressant, making it potentially useful in the treatment of organ transplant rejection and various autoimmune diseases. The precise mechanism by which SFA operates is not known, but it appears to function in a manner distinctly different from other immunosuppressants such as FK506 and rapamycin. Preliminary studies have shown that that SFA forms a complex with cyclophilin A (CypA), which then acts on an unknown target(s). The working hypothesis of this proposal is that SFA acts on a previously unknown member of the signal transduction pathway leading to T cell activation. This hypothesis will be tested by: (1) sequencing a BAG clone containing the SFA biosynthetic cluster to obtain insights into the biosynthesis of the molecule;(2) preparing a biotin-tagged SFA derivative;and (3) incubating the biotinylated probe with a cell extract, then purifying and analyzing the protein mixture to identify the target of SFA (TOS). Identifying the biological target of the SFA-CypA complex will shed light on the mechanism of action of this novel immunosuppressant and enrich our understanding of the immune system. In long-term research based on this work, the structure of SFA may well present a new scaffold upon which to engineer biological probes to study the immune system and improve human health. PUBLIC HEALTH RELEVANCE: Sanglifehrin A (SFA) is a recently discovered natural compound that has strong biological activity as an immunosuppressant, making it potentially useful in the treatment of organ transplant rejection and various autoimmune diseases. Little is known about how SFA operates in the body, but it appears to have an activity distinctly different from other immunosuppressants currently used in medicine. This study aims to elucidate the mechanism by which SFA functions, enriching our understanding of the immune system and this novel therapeutic agent.